The prevention of home-cage grid climbing affects muscle strength in mice

Experimenters and treatment methods are the major contributors to data variability in behavioral neuroscience. However, home cage characteristics are likely associated with data variability. Mice housed in breeding cages spontaneously exhibit behavioral patterns such as biting into the wire grid and climbing on the grid lid. We aimed to clarify the effect of covering the stainless steel wire grid lid in commonly used home cage with Plexiglas to prevent climbing on muscle strength in mice. Furthermore, we investigated the effects of climbing prevention on activity and anxiety-like behavior, and the impact of climbing prevention during the postnatal development period and adulthood on muscle strength. Muscle strength, anxiety-like behavior, and locomotor activity were assessed by a battery of tests (wire hang, suspension, grip strength, rotarod, elevated-plus maze, and open field tests). Mice prevented from climbing the wire grid during postnatal development displayed lower muscle strength than those able to climb. Moreover, mice prevented from climbing for 3 weeks following maturity had weakened muscles. The muscle strength was decreased with 3 weeks of climbing prevention in even 1-year-old mice. In summary, the stainless steel wire grid in the home cage contributed to the development and maintenance of muscle strength in mice.


Wire hang test.
For the wire hang test, mice were placed on a wire mesh that was subsequently inverted and gently waved, forcing the mice to grip the wire. We recorded the latency to fall. A wire hang test apparatus (O'Hara & Co., Tokyo, Japan) was used for this test.

Wire suspension test.
For the wire suspension test, we placed a 2-mm-thick wire parallel to the floor and secured it approximately 40 cm above a layer of bedding. We let the mice grasp the wire with their forepaws only; we lowered the hindlimbs so that mice hung from the wire only by their forepaws. We recorded the latency to fall 24 .
Grip strength test. We used a grip strength meter to assess the forelimb grip strength. Mice were lifted and held by their tail such that their forepaws could grasp a wire grid; subsequently, they were gently pulled backwards until they released the grid. We recorded the peak force applied by the mouse's forelimbs in centinewtons (cNs).

Rotarod test.
We assessed motor coordination and balance using the rotarod test 25 . This test used an accelerating rotarod (RTR-M5; Melquest, Toyama, Japan). Mice were placed on a rotating drum (3.9-cm diameter) and the time they were able to maintain balance on this rod was measure. The speed of the rotarod was accelerated from 4 to 40 rpm over a period of 5 min. The intertrial interval for this test was 20 min. All mice were subjected to the test without any pre-test training.
Hot plate test. We used the hot plate test to evaluate nociception (sensitivity to a painful stimulus) 26

Open field test.
We used the open field test to examine exploratory behavior, anxiety-like behavior, and general locomotor activity 17,30 . Each mouse was placed in the center of an apparatus comprising a square area surrounded by white acrylic walls (45 × 45 × 40 cm) 23 . We recorded the total distance travelled (m), number of entries into the central area, and time spent in the central area (s). The central area was defined as the middle 20 × 20 cm area of the field. The test chamber was illuminated at 100 lx. Data were collected over 20 min. Data analysis was performed using ANY-MAZE software.
Wire grid climbing measurement. For measurements of wire grid climbing, mice were acclimated to the single housing environment, and their behavior was monitored from 14:00 to 11:00 the day after acclimation. Images were captured via a video camera, and we measured the number of episodes and amount of time climbing on the wire grid. We used 4-week-(n = 4), 10-week-(n = 4), and 1-year-old (n = 4) mice for this test.
Statistical analyses. Statistical analyses were performed using SPSS software (IBM Corp, Tokyo, Japan).
Data were analyzed using the one-way analyses of variance (ANOVA), followed by Tukey's test, or two-way repeated-measures ANOVA, followed by Fisher's least significant difference test. P-values < 0.05 were considered statistically significant. All data are presented as box plots or the mean ± standard error.
Ethics approval and consent to participate. All

Results
Effects of grid-climbing prevention on physical characteristics. There were no significant differences in body weight gain between the climbing and non-climbing mice (Fig. 3, F1,224 = 0.158, p = 0.692). In the wire hang test, the latency to fall was significantly lower in the non-climbing mice than in the climbing mice ( Fig. 4A

Effects of grid-climbing prevention on the elevated plus-maze test.
In the elevated plus-maze test, there were no significant differences between climbing and non-climbing mice in the total distance travelled (

Effects of grid-climbing prevention on the open field test.
In the open-field test, there were no significant differences between climbing and non-climbing mice in the distance travelled ( Wire grid climbing in the home cage. Additionally, we examined grid climbing behavior in the home cage in 4-week-, 10-week-, and 1-year-old mice. There was no significant difference between 4-week-and 10-week-old mice in the total number of gird-climbing episodes (Fig. 8A, F2,9 = 4.379, p = 0.047). However, the number of grid-climbing episodes was significantly lower in 1-year-old mice than in 10-week-old mice. Additionally, the total time spent grid climbing was significantly lower in 1-year-old mice than in both 4-week-and 10-week-old mice (Fig. 8B, F2,9 = 9.162, p = 0.007). There was no significant difference between 4-week-and 10-week-old mice in the total time spent grid climbing. The mean time per grid-climbing episode was significantly lower in 1-year-old mice in both 4-week-and 10-week-old mice (Fig. 8C, F2,9 = 10.209, p = 0.005). There was no significant difference between 4-week-and 10-week-old mice in the mean time per grid-climbing episode. We plotted graphs for the number of gird-climbing episodes in each 30 min period (Fig. 8D), time spent grid climbing during each period (Fig. 8E), and mean time grid climbing during each period (Fig. 8F). High climbing activity was observed in the early dark phase. www.nature.com/scientificreports/ Effect of grid-climbing prevention in middle-aged mice. We examined the effects of grid-climbing prevention in weight-matched (within 2 g) 1-year-old mice. Following climbing prevention for 3 weeks, we examined their neuromuscular strength. There were no significant differences between climbing and non-climbing 1-year-old mice in the wire hang test (Fig. 9A, F1,13 = 0.132, p = 0.722) and wire suspension test (Fig. 9B, F1,13 = 3.527, p = 0.083). Grip strength was significantly lower in 1-year-old non-climbing mice than in 1-yearold climbing mice (Fig. 9C, F1,13 = 5.181, p = 0.040).
Effects of promoted grid climbing in adulthood. Subsequently, we examined the effects of promoted grid climbing in mature mice (Fig. 10A). We raised the position of the food pellets in the home cage to facilitate grid climbing. Following 3 weeks of treatment, we examined their neuromuscular strength. There were no differences between climbing and promoted-climbing mice in the latency to fall in the wire hang test (Fig. 10B, F1,18 = 0.734, p = 0.403) and wire suspension test (Fig. 10C, F1,18 = 0.004, p = 0.948), and in grip strength test (Fig. 10D, F1,18 = 0.286, p = 0.600).

Discussion
The purpose of the present study was to investigate the effect of preventing climbing on the stainless steel wire bars in the lid of commonly used cages on the muscle strength of mice. We found that stainless steel wire lids in the home cage contributed to the development and maintenance of muscle strength in mice.
Mice that were prevented from climbing the grid lid in their home cage from post-weaning to maturity did not demonstrate a difference in body weight gain compared to those that were allowed to climb. We had predicted weight loss with climbing prevention because of stress. Chronic stress reportedly reduces body weight in mice 31,32 . However, the present results suggest that grid-climbing prevention was not a chronic stress for the mice. Furthermore, there were no changes in activity level with grid-climbing prevention, as indicated by the www.nature.com/scientificreports/ elevated plus-maze and open field test results. Considered together, these results suggest that the energy level in the home cage did not change despite the prevention of grid climbing in the home cage. In order to further clarify the presence or absence of stress with climbing prevention, it is necessary to measure stress hormones and the amount of food consumption in future studies. Mice housed in breeding cages voluntarily perform several behavioral patterns, including bar gnawing, bar swiveling, and bar jumping, all of which are involved in climbing the grid cage lid 12 . These interactions with the steel bars present in the cage lid represent key elements of behavior in the home cage of laboratory mice 33 ; however, there are no reports on their association with muscle strength. Researchers have reported that the forelimb grip strength in mice is maximal at 4 months, decreases at 7 months, and is stable until 2 years of age 34 . In contrast, the hindlimb grip strength significantly increases from 4 to 7 months, and decreases at 18 months of age. Researchers have not investigated the relationship between grip strength and the lid characteristics of the home cage. In the present study, mice that were prevented from climbing the wire grid lid from post-weaning to maturity demonstrated a marked decrease in muscle strength compared to that in mice that were allowed to climb, as assessed by the wire hang and grip strength tests. In the wire suspension test, mice that were prevented from climbing the grid lid did not hold the wire in their forelimbs, but instead held it with their armpits. This behavior indicated that there was no habit of grabbing the wire with the forelimbs or that the forelimbs were significantly weak. Accordingly, the lid grid of the home cage promoted the development of muscle strength in developing mice. This necessitates further research to investigate the essential muscle development of mice and the period of development.
Motor coordination in mice has traditionally been assessed by the rotarod test 35,36 . In this test, mice must remain upright and walk forward to avoid falling. It is a sensitive, objective, and quantifiable test. Rodents with   www.nature.com/scientificreports/ rotarod test. Thus, lid grid climbing in the home cage provided an opportunity for developing mice to cultivate athletic performance. Mice that were prevented from grid climbing and then subsequently housed in cages allowing grid climbing for 3 weeks did not have similar strength to control mice. However, we did not compare the strength of these mice initially to those with continued grid climbing prevention; accordingly, it is unknown whether muscle www.nature.com/scientificreports/ strength was partially restored. It is possible that these mice did not develop the habit of climbing the lid grid, thus further research is necessary to clarify the findings. All of the mice (regardless of condition) tended to be less active in the elevated plus maze test than in the open field test, suggesting that they had anxiety for heights. It is also possible that the mice behaved differently in low light. Following weaning, mice that were prevented from grid climbing did not demonstrate changes in their activity or anxiety-like behavior in the elevated plus maze and open field tests. Preventing climbing behavior in the home cage for only 4 weeks following birth has been shown to cause behavioral abnormalities in female mice 13 . However, the prevention of climbing behavior did not cause anxiety-like behavior in the male mice used in the present study. This suggests that grid-climbing prevention may not affect the psychological behavior of male mice. Further research is required to directly compare the effect of grid-climbing prevention on anxietylike behavior in male and female mice.
In the hot plate test, mice prevented from grid climbing did not show a change in sensitivity to temperature. Hot plate tests are commonly used to assess heat pain susceptibility 37 . The present results demonstrate that gridclimbing prevention does not exert an effect on the development of nociception in mice.
In the present study, the number and time of grid-climbing episodes depended on the age of the mouse. Furthermore, we found that 1-year-old mice also exhibited effects of grid-climbing prevention on muscle strength. Climbing behavior in the home cage is known to vary with age, sex, and tension 38 . In a previous study of 1-, 2-, 3-, 4-, 5-, and 6-month-old mice, the maximum climbing time was observed at 2 months of age 38 , consistent with the present results. A decline in climbing behavior with aging is associated with a decline in overall activity and exploratory drive with aging 39,40 . The present findings demonstrated that climbing behavior in the home cage is required for the development and maintenance of muscle strength and motor ability in mice, even in 1-year-old mice. Further research is needed to determine whether increasing exploratory behavior in the home cage increases climbing behavior and maintains muscle strength.
In the wire hang and grip strength tests, mature mice displayed a decrease in muscle strength after gridclimbing prevention for 3 weeks compared to that in control mice. Peak climbing behavior is observed at approximately 2 months following birth. As mice exhibit climbing behavior throughout their lives 39,40 , muscle strength can be maintained by climbing the grid lid in the home cage. Climbing prevention in 10-week-old mice has been reported to result in decreased muscle strength in grip strength tests 41 . The observed decrease in muscle strength indicated that muscles were weakened by the prevention of grid climbing. Furthermore, 3 weeks of prevention was sufficient to induce muscle weakness. Further research is warranted to determine whether the duration of prevention and age of mice exert different effects on muscle weakness. Additionally further studies are needed to obtain detailed data on muscle mass and muscle atrophy.
In 1-year-old mice, preventing grid climbing for 3 weeks did not result in any change in the wire hang and wire suspension tests; nonetheless, it resulted in a decrease in grip strength. These results indicate that middle-aged mice prevented from grid climbing have decreased forelimb grip strength. The grip strength of forelimbs in mice remains stable after 7 months of age; however, that of the hindlimbs decreases at 18 months following birth 32 . In the present study, 1-year-old mice exhibited climbing behavior in their home cage. This climbing behavior in the home cage was associated with the maintenance of forelimb grip strength in mice at up to 12 months of age. To prevent mouse habituation to the grip strength test, we did not perform the grip strength test prior to grid-climbing prevention. Therefore, it is possible that there was a pre-existing difference in the grip strength in the mice.
In the present study, mice housed in home cages with elevated food to promote climbing did not display increased muscle strength. Mice frequently hang in their home cage 38 . It is unclear if the number and time of grid-climbing episodes were increased in the home cage environment used in this experiment; however, it did not lead to further muscle strengthening. Thus, grid climbing in normal breeding cages is sufficient for maintaining muscle strength in mice.
Grid bar-related activities have been described as stereotypes owing to their repetitive and invariant nature; however, this activity should merely be considered as a form of exercise or behavioral habit 42,43 . On the hand, grid bar-related activities may represent an attempt to leave the home cage and explore the external environment 12 . The role of grid bar-related activities in representing purposeless or functional behavior remains debatable. The present experimental results did not address this argument. However, the climbing behavior of mice in the home cage can be effectively used for pain assays 38 . The present findings revealed that the climbing behavior with respect to the grid bar promoted the development of muscle strength in mice, and was related to its maintenance.
The early post-weaning period is sensitive to environmental effects on the rodent brain 44 . Changes in housing conditions during this period exert long-term effects on both brain and behavior 45 . Environmental enrichment is a widely applied term and includes an experimental paradigm in which intensive environmental enrichment strategies are used to investigate the impact of complex environments 46 . This environmental enrichment causes changes in mouse behavior 47 , emotions 48,49 , physiology 50 , and neurobiology 51,52 , compared to that with normal simple cages 53 . However, the direction and magnitude of the effect of environmental enrichment depends on the type and combination of enrichment 54 , mice strain 55 , sex 56 , time and duration of enrichment 57 , and parameters studied 58 . Researchers have not yet comprehensively investigated the effects of the shape and material of the lid in the home cage on mouse behavior. Mice climb the lid and roam through the home cage 59 . Moreover, they gnaw on the wire grid on the lid 60 . The present findings suggest that the mice climbed the wire grid lid to develop and maintain their muscle strength. Therefore, the wire grid lid of the home cage also contributes to environmental enrichment.
Muscle weakness is an important phenotype of several diseases associated with movement disorders and increased mortality. The forces generated by a muscle are largely determined by the size of the muscle, type of fiber, and coupling process of excitation and contraction. The strength of the limbs of experimental mice can be evaluated using a commercially available grip strength meter. This test is a widely used non-invasive method, www.nature.com/scientificreports/ specifically used to assess the strength of the forelimbs and/or hindlimbs of mice to assess effects of disease (e.g., cancer, dystrophy) or drug treatment on skeletal muscle 61,62 . Wire climbing is ideal for measuring the coordination and endurance of mice muscles 63,64 . Moreover, it has been applied to assess the grip strength, balance, and endurance in mice following cerebral infarction 65 . However, the relationship between the lid of the home cage and muscle strength has not been previously reported. The present findings indicate that previous reports on mouse muscle weakness may be affected by the shape of the lid and the exploratory behavior of mice in their home cage. For example, mice may improve their muscle strength by frequently climbing the grid lid in the home cage, despite genetic modification to reduce their muscle strength. Researchers should pay attention to the cage lid upon conducting experiments on mouse muscle strength. In addition, a description of the lid of the home cage is required in the "Materials and methods" section. The present results were obtained using only male mice. Climbing behavior in the home cage depends on age and sex 38 , and its effect on the muscle strength of mice may differ. This warrants further research using female mice.

Conclusion
The wire grid on the lid of the home cage contributed to the development and maintenance of mouse muscle strength. Researchers should pay attention to the shape and material of the mouse cage lid.

Data availability
The dataset is available on reasonable request from the corresponding author.